The metal ion copper (Cu) is essential for life and plays critical roles as a cofactor for a wide range of human biochemical process including energy generation, neuropeptide maturation, reactive oxygen detoxification, iron absorption and connective tissue formation. Mutations in genes encoding proteins involved in Cu acquisition, utilization or detoxification cause severe human disease and abnormal Cu homeostasis is also associated with Alzheimer's Disease, immune system dysfunction and other pathophysiological states. Moreover, Cu resistance has been implicated as an important mechanism in microbial virulence. While many of the proteins involved in Cu import, intracellular delivery and efflux have been identified in model systems such as the baker's yeast, and homologues found in humans, there are many gaps in our knowledge of the components involved in Cu homeostasis and the adaptive responses to Cu deficiency or Cu toxicity. Cryptococcus neoformans is a fungal pathogen that is acquired by the respiratory route and disseminates to the brain, where it causes meningitis and is responsible for ~600,000 human deaths/year. In contrast to other yeast model systems such as the baker's yeast, C. neoformans preferentially utilizes Cu-dependent respiration, rather than fermentation for energy generation, and has several additional Cu-dependent enzymes such as amine oxidase, laccase and glyoxal oxidase that demonstrate a more extensive Cu biology and suggest added complexity in Cu homeostasis. We demonstrated that C. neoformans robustly regulates the transcription of an unprecedented number of genes in response to Cu deficiency or Cu excess, via the direct action of the Cuf1 Cu-responsive transcription factor. Given that all fungal genes regulated by Cu have been previously demonstrated to encode proteins that function in Cu homeostasis, or utilize Cu, studies in C. neoformans have a high probability of identifying new Cu homeostasis genes and giving new insights into the biology of Cu. Moreover, since recent studies demonstrate that Cuf1 is important for a lethal C. neoformans infection in mice, understanding which Cuf1-regulated Cu homeostasis genes function in fungal virulence has direct relevance to human health. We outline three Specific Aims, using genetics, biochemistry, cell and molecular biology in C. neoformans to identify novel Cu homeostasis genes and their role in the biology of Cu. Furthermore, we will use mouse models of C. neoformans infection to decipher the roles of established and novel components of the Cu homeostasis machinery in virulence. Given the critical roles for Cu in human health, and the requirement for Cu homeostasis in fungal virulence, these studies aim to further our understanding of Cu acquisition and detoxification in eukaryotic cells and understand the role of Cu in lethal fungal meningitis.